Container for Nucleic Acid Extraction, Method of Cleaning Solid Matrix and Relevant Cleaning Mechanism, and Method of Purifying Nucleic Acid

ABSTRACT

The present invention relates to a nucleic acid extraction container ( 2 ) which includes a nucleic acid extraction element ( 20 ) for extracting a target nucleic acid from a sample and supporting the extracted nucleic acid, and a container main body formed as a separate member from the nucleic acid extraction element and having a having a housing vessel ( 27 ) for storing the nucleic acid extraction element ( 20 ). The nucleic acid extraction element ( 20 ) includes, for example, a solid matrix ( 23 ) for supporting the target nucleic acid, and a holding member ( 20 ) for holding the solid matrix ( 23 ). Preferably, the solid matrix ( 23 ) is held as tilted with respect to a vertical axis of the holding member ( 20 ).

TECHNICAL FIELD

The present invention relates to a technique for extracting a targetnucleic acid from a sample.

BACKGROUND ART

Analysis of nucleic acid has been playing an important role in themedical field in diagnosis of infectious or genetic diseases on agenetic level. Today, its field of application is expanding beyond themedical field into various other fields such as agriculture and food.Generally, analysis of nucleic acid includes processes of extraction ofnucleic acid from a sample, amplification of the purified nucleic acid,and detection of the amplified nucleic acid. When personnel cost,repeatability and analysis efficiency are considered, it is preferablethat each of the processes is performed automatically by a machine, andideally all the processes should be performed automatically by a machine(See Patent Documents 1 and 2 for example).

In an effort toward automated purification of nucleic acid, methodswhich use a nucleic acid-binding carrier have been developed. As anexample of such methods, there is a method which utilizes nucleicacid-binding silica particles and a chaotropic ions (See Patent Document3 for example). This method includes mixing a sample with nucleicacid-binding silica particles and chaotropic ions, which are capable ofisolating nucleic acid in the sample, in order to bind the nucleic acidin the sample with the nucleic acid-binding silica particles, thenseparating the sample into solid-phase and liquid-phase, and theneluting the nucleic acid which is bound to the nucleic acid-bindingsilica particles. However, separating solid-phase from liquid-phaserequires some operation such as centrifugation and filtration using afilter, which leads to complicated operation procedure in the apparatusas well as complicated configuration of the automated apparatus.

Another example of the method of using a nucleic acid-binding carrier isone which uses a magnetic carrier (See Patent Document 4 and 5 forexample). This method includes absorption of nucleic acid by magnetizedsilica particles, then separating the silica particles with a magnet,then cluting the nucleic acid from the separated silica particles, andthen recovering the eluent. This method is advantageous for automatedapparatus since solid-phase and liquid-phase are separated from eachother without e.g. centrifugal separation.

Problems of this method, however, include a relatively low recovery rateof the nucleic acid and that the rate itself is subject to influencefrom the kind of specimen. Another problem is that magnetized silicaparticles act as an inhibitor in an amplification reaction when PCR(Polymerase Chain Reaction) method is employed as a method of amplifyingthe nucleic acid. In addition, magnetized silica particles are not verymuch compatible with a typical detection method for nucleic acid inwhich marking is made to the nucleic acid and the marks are opticallymeasured. If this method is used, the magnetized silica-particles causemeasurement errors, and variation of the amount of magnetized silicaparticles at the time of purification lower repeatability.

Still another example of the method of purifying nucleic acid is oneusing a solid matrix which is impregnated with e.g. reagents necessaryfor extracting nucleic acid (See Patent Documents 6 through 8 forexample). In this method, a target nucleic acid is extracted by drippinga sample onto the solid matrix. Thereafter, cleansing is performed toremove extra components which may act as inhibitors in the amplificationprocess of the nucleic acid. More specifically, cleansing off of theextra components is performed by punching a disc out of the solidmatrix, placing the punched discal solid matrix into a microtube, andeluting the extra components from the discal solid matrix in themicrotube.

If this method is employed, a step of punching a part of the solidmatrix is required, and further, in order for the solid matrix to bepunched, drying the solid matrix is required in order to punch the solidmatrix. Therefore, this method requires considerable time before thediscal solid matrix can be cleansed. Also, since the method of cleansingof the discal solid matrix is performed by diffusing the extracomponents from the solid matrix into the cleansing liquid, this methodrequires a considerable amount of time for cleansing. Especially, inorder to achieve the cleansing to a sufficient level, it is necessary tochange the cleansing liquid held in the microtube repeatedly, which alsoextends the time necessary for the cleansing. Moreover, to shorten thetime required for cleansing, the size of the discal solidmatrix has tobe reduced, whereby the amount of target nucleic acid recoverable fromthe sample becomes small. As a result, sensitivity in the nucleic aciddetection process is expected to be low and therefore, the time foramplification need be increased in order to obtain a sufficient amountof nucleic acid.

Still further, in the method of using a discal solidmatrix, theamplification of the nucleic acid is performed in the microtube used forcleansing the nucleic acid, and then, the analysis of the nucleic acidis also made in the microtube with an optical technique. Therefore, ifthe discal solid matrix is unduly large, the discal solid matrix blocksthe optical path for the photometry, making impossible to analyze thenucleic acid. In order to avoid such a problem as this, the discal solidmatrix is required to be punched into a sufficiently small size, forexample, 2 mm, which leads to the above-described problem that theamount of the target nucleic acid recoverable from the sample isdecreased. In addition, punching a disc of solid matrix which is notgreater than 2 mm in size poses a big burden on the user, and thepunching operation will not be an efficient process.

Automated apparatus for amplification of nucleic acid using the PCRmethod is in advancement, and there is already PCR apparatuses availableon the market. A popular PCR apparatus can perform amplification ofnucleic acid and detection of the amplified nucleic acid.

However, PCR apparatuses available on the market mustis required to beused with an amplification kit dedicated to the apparatus. Typically,the amplification kit includes a lid and a container which contains somereagents a container which has a lid and pre-filled with reagents suchas a primer and polymerase. Therefore, the user is forced to open thelid of the container first, then dispense pour the nucleic acid solutioninto the container, stir the reaction liquid in the container, close thelid, and then set the container to the PCR apparatus. In other words,the common amplification kit depends heavily on the user's manualoperation, which poses a burden upon the user. In addition, due to themajor manual intervention by the user, analysis efficiency is low, andthere is room for concern for decreased repeatability due to differentlevels of skill among the users. Further, since the container used forthe amplification kit is a general-purpose product manufactured by resininjection molding, integrally with the lid by resin molding, so it isdifficult to design for the PCR apparatus capable of opening and closingto open or close the lid automatically. For this reason, it is difficultin for PCR apparatuses to replace the user's manipulation to performoperations automatically with the method using a common dedicated kit.

Other examples of the method of amplifying nucleic acid, other than PCRmethod include ICAN (Isothermal and Chimeric Primer-initiatedAmplification of Nucleic acid) method and LAMP (Loop-Mediated IsothermalAmplification) method. In comparison to the PCR method, these methodsrequire a sufficiently higher level of purification of nucleic acid inorder to achieve sufficient amplification of a target nucleic acid.Therefore, if purification and amplification of nucleic acid are to beautomated using an amplification method other than PCR method, atechnique for increasing the level of nucleic acid purification must beestablished.

Patent Document 1: JP-A 2001-149097 Gazette

Patent Document 2: JP-A 2003-304899 Gazette

Patent Document 3: JP Patent 2680462 Gazette

Patent Document 4: JP-A S60-1564 Gazette

Patent Document 5: JP-A H9-19292 Gazette

Patent Document 6: U.S.A. Pat. No. 5,496,562 Specification

Patent Document7: U.S.A. Pat. No. 5,939,259 Specification

Patent Document8: U.S.A. Pat. No. 6,168,922 Specification

DISCLOSURE OF THE INVENTION

The present invention aims at mechanical automation for the entiresequence of nucleic acid analysis including purification of nucleicacid, amplification of the nucleic acid and measurement of the nucleicacid, thereby reducing burden on the user and improving analysisefficiency and repeatability.

Further, the present invention aims at providing a nucleic acidpurification technique which allows a variety of amplification methodsapplicable to an automated purification and amplification of nucleicacid by an apparatus.

A first aspect of the present invention provides a nucleic acidextraction container which includes: a nucleic acid extraction elementfor extraction of a target nucleic acid from a sample and carrying ofthe extracted nucleic acid; and a container main body formed as aseparate member from the nucleic acid extraction element, having ahousing vessel for storage of the nucleic acid extraction element.

Preferably, the nucleic acid extraction element includes a solid matrixfor supporting the target nucleic acid, and a holding member for holdingthe solid matrix.

The solid matrix is held as tilted with respect to a vertical axis ofthe holding member for example, and preferably held horizontally orsubstantially horizontally with respect to the vertical axis. In thiscase, the solid matrix is preferably formed as a disc.

The state in which the solid matrix is held as tilted with respect tothe vertical axis can be achieved by e.g. holding the solid matrix aspenetrated by the holding member. In this case, the holding member isformed to include for example: a tapered portion with a decreasingdiameter toward a tip; a pin-shaped portion extending from the taperedportion for penetration through the solid matrix; and a holding tab forpreventing the solid matrix from coming off the pin-shaped portion.

Alternatively, the solid matrix may be held in parallel to orsubstantially in parallel to the vertical axis of the holding member. Inthis case, the solid matrix is preferably formed as a sheet.

The state in which the solid matrix is parallel to or substantiallyparallel to the vertical axis can be achieved by suspending the solidmatrix with the holding member for example. In this case, the holdingmember is formed to include a clip portion, for example, for clipping anend of the solid matrix thereby suspending the solid matrix.

It is preferable that the container main body includes one or morecleansing vessels for holding cleansing liquid necessary to remove anextra component from the solid matrix. In this case, the container mainbody is preferably provided with surplus cleansing liquid removal meansfor removal of surplus cleansing liquid from or around the solid matrix.

The surplus cleansing liquid removal means includes a water absorbingmember for example. The water absorbing member may be of a porousmaterial such as resin foam and cloth.

The nucleic acid extraction container according to the present inventionis provided as a cartridge for example, for setting in a nucleic acidanalyzer.

If the nucleic acid analyzer includes a carrier member for taking thenucleic acid extraction element, which supports the target nucleic acid,from the housing vessel and moving the nucleic acid extraction elementto another location, the nucleic acid extraction element is formed toinclude an engagement portion for engagement with the carrier member. Inthis case, the engagement portion is formed to be cylindrical forexample, for fitting with a tip portion of the carrier member, andpreferably, the engagement portion includes one or more cutouts forapplication of elastic force to the carrier member upon fitting with thetip portion of the carrier member. The cutout or cutouts include atleast one of a slit, a notch and a through hole.

On the other hand, the holding member includes a projection, forexample, which is used for breaking the fit between the carrier memberand the engagement portion. Preferably, the projection is provided by aflange extending outward. In this case, the housing vessel is formedwith a stepped portion for seating the projection upon placement of thenucleic acid extraction element in the housing vessel.

A second aspect of the present invention provides a method of removingan extra component other than a target nucleic acid from a solid matrixwhich supports the target nucleic acid, using a cleansing liquid. Themethod includes a repeated cycle of a state in which the solid matrix issubmerged in the cleansing liquid and a state in which the solid matrixis not submerged in the cleansing liquid, caused by moving the solidmatrix in up-and-down directions relatively to the cleansing liquid.

In the repeated cycle of the state in which the solid matrix issubmerged in the cleansing liquid and the state in which the solidmatrix is not submerged in the cleansing liquid, the solid matrix istilted with respect to the up-and-down directions. Preferably, the solidmatrix is perpendicular or substantially perpendicular to theup-and-down directions.

A third aspect of the present invention provides a method of purifying atarget nucleic acid using a nucleic acid extraction element whichincludes a solid matrix for supporting the target nucleic acid and aholding member for holding the solid matrix. The method includes: anucleic acid supporting step of causing the solid matrix to support thetarget nucleic acid in a sample by soaking the solid matrix in thesample; and a cleansing step of removing an extra component other thanthe target nucleic acid from the solid matrix, using a cleansing liquid.The cleansing step is provided by a repeated cycle of a state in whichthe solid matrix is submerged in the cleansing liquid and a state inwhich the solid matrix is not submerged in the cleansing liquid, causedby moving the solid matrix in up-and-down directions relatively to thecleansing liquid.

In the method of purifying nucleic acid according to the presentinvention, the solid matrix held by the holding member is tilted withrespect to a vertical axis of the holding member in the nucleic acidextraction element. In this case, the cleansing step is performed bymoving the extraction element in up-and-down directions, with the solidmatrix tilted with respect to the up-and-down directions. Preferably,the cleansing step is performed by moving the nucleic acid extractionelement in the up-and-down directions, with the solid matrix beingperpendicular or substantially perpendicular to the up-and-downdirections.

The cleansing step includes removal of surplus cleansing liquid from thesolid matrix by a water absorbing member after completion of therepeated soaking of the solid matrix in the cleansing liquid.

A fourth aspect of the present invention provides a mechanism forremoving an extra component other than a target nucleic acid from asolid matrix which supports the target nucleic acid, using a cleansingliquid. The mechanism repeats a cycle of a state in which the solidmatrix is submerged in the cleansing liquid and a state in which thesolid matrix is not submerged in the cleansing liquid, by moving thesolid matrix in up-and-down directions relatively to the cleansingliquid.

The cleansing mechanism according to the present invention moves thesolid matrix as tilted with respect to the up-and-down directions forexample. Preferably, the solid matrix is perpendicular or substantiallyperpendicular to the up-and-down directions when moved in theup-and-down directions.

It should be noted here that the term “sample” used in the presentinvention includes biologic samples (such as whole blood, blood serum,blood plasma, urea, saliva and bodily fluid) from animals and nonanimals. The term “nucleic acid” means DNA or RNA, including duplex DNA,uniplex DNA, plasmid DNA, genomic DNA, cDNA, exogenous RNA parasiticorganism (such as virus, bacillus and fungus) and endogenous RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overall perspective view for describing a nucleic acidanalyzer.

FIG. 2 is a plan view showing an internal configuration of the nucleicacid analyzer in FIG. 1.

FIG. 3 is a sectional view taken in lines III-III in FIG. 2.

FIG. 4 is a sectional view taken in lines IV-IV in FIG.

FIG. 5 is an overall perspective view showing a nucleic acidpurification cartridge.

FIG. 6 is a sectional view taken in lines VI-VI in FIG. 5.

FIG. 7A is an overall perspective view of a nucleic acid extractionelement in the nucleic acid purification cartridge, and FIG. 7B is asectional view of the nucleic acid extraction element.

FIG. 8 is an overall perspective view of a nucleic acid amplificationcartridge.

FIG. 9 is a sectional view taken in lines IX-IX in FIG. 8.

FIG. 10 is a sectional view of a primary portion for describing a solidmatrix cleansing operation.

FIG. 11 is a sectional view of a primary portion showing a lid removingoperation from the nucleic acid amplification cartridge.

FIG. 12 is a sectional view of a primary portion for describing aremoving operation of a diffused nucleic acid extraction element using alid.

FIG. 13A is a sectional view of a primary portion for describing aplacing operation of the nucleic acid extraction element into a reactionvessel or the nucleic acid amplification cartridge, and FIG. 13B is asectional view of a primary portion for describing a lid removingoperation from the reaction vessel.

FIG. 14 is a sectional view taken in lines XIV-XIV in FIG. 13.

FIG. 15 is a sectional view representing a section taken in lines XV-XVin FIG. 2, for describing a temperature controlling mechanism and ameasurement mechanism.

FIG. 16 is a plan view showing an internal configuration, for describinga nucleic acid analyzer.

FIG. 17 is a sectional view taken in lines XVII-XVII in FIG. 16.

FIG. 18 is a sectional view taken in lines XVIII-XVIII in FIG. 16.

FIG. 19 is an overall perspective view of a nucleic acid purificationcartridge.

FIG. 20A is a perspective view showing a nucleic acid extraction elementin the nucleic acid purification cartridge,

FIG. 20B is a plan view thereof, and FIG. 20C is a sectional view takenin lines XXc-XXc in FIG. 20A.

FIG. 21 is a sectional view of a container of the nucleic acidpurification cartridge, representing a section taken in lines XXI-XXI inFIG. 19.

FIG. 22 is a sectional view of a primary portion showing a removaloperation of the nucleic acid extraction element from a housing vesselin the container.

FIG. 23 is an overall perspective view of a nucleic acid amplificationcartridge.

FIG. 24A is a sectional view taken in lines XXIVa-XXIVa in FIG. 23, andFIG. 24B is a sectional view showing a state where a lid is removed inFIG. 24A.

FIG. 25 is a front view of a primary portion, for describing anoperation of attaching a tip to a nozzle.

FIG. 26 is a front view of a primary portion, for describing anoperation of attaching the nucleic acid extraction element to thenozzle.

FIG. 27 is a front view of a primary portion, for describing anoperation to detach the tip from the nozzle.

FIG. 28 is a front view of a primary portion, for describing anoperation of detaching the nucleic acid extraction element from thenozzle.

FIG. 29 is a sectional view of a primary portion, showing an operationof inserting a turning member into the lid of the nucleic acidamplification cartridge.

FIG. 30 is a sectional view of a primary portion, showing an operationof removing the lid of the nucleic acid amplification cartridge.

FIG. 31 is a sectional view of a primary portion showing an operation ofputting the nucleic acid extraction element into a reaction vessel inthe nucleic acid amplification cartridge.

FIG. 32 is a sectional view of a primary portion for describing anoperation of re-attaching the lid of the nucleic acid amplificationcartridge.

FIG. 33 is a sectional view, representing a section taken in linesXXXIII-XXXIII in FIG. 16, for describing the measurement mechanism.

FIG. 34 is a graph showing a result of fluorescence intensitymeasurement according to Embodiment 1 (PCR method), with the horizontalaxis representing temperatures and the vertical axis representingderivative values of the fluorescence intensity.

FIG. 35 is a graph showing a result of fluorescence intensitymeasurement according to Embodiment 2 (ICAN method), with the horizontalaxis representing the number of cycles and the vertical axisrepresenting fluorescence intensity.

FIG. 36 is a graph showing a result of fluorescence intensitymeasurement according to Embodiment 3 (LAMP method), with the horizontalaxis representing the number of cycles and the vertical axisrepresenting fluorescence intensity.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, description will be made for a first and a secondembodiments of the present invention with reference to the drawings.

First, the first embodiment will be covered with reference to FIG. 1through FIG. 15.

FIG. 1 through FIG. 4 show a nucleic acid analyzer 1 which automaticallyperforms extraction of nucleic acid in a sample, amplification of theextracted nucleic acid and analysis of the amplified nucleic acid. Asshown in FIG. 1 and FIG. 2, a plurality of nucleic acid purificationcartridges 2 and the same number of nucleic acid amplificationcartridges 3 are set in a case 10 when the analyzer is used.

As shown in FIG. 5 and FIG. 6, the nucleic acid purification cartridge2, which is a component that enables automatic purification of nucleicacid, includes a nucleic acid extraction element 20 and a cartridge mainbody 21.

The nucleic acid extraction element 20, which is used for extractingnucleic acid from the sample, is housed in a housing vessel 27of thecartridge main body 21 to be described later. As shown clearly in FIG.7A and FIG. 7B, the nucleic acid extraction element 20 has a holdingmember 22 and a solid matrix 23.

The holding member 22 includes a cylindrical part 24, a flange 25, andholding portion 26, and is formed entirely by resin molding for example.

The cylindrical part 24 serves to move the nucleic acid extractionelement 20 (See FIG. 4 and FIG. 12), and has a recess 24A and an alatching head 24B. The recess 24A serves to accommodate an insertion pin50 of a nucleic acid purification mechanism 5 or a pin 36B of a lid 31of the nucleic acid amplification cartridge 3 (See FIG. 4 and FIG. 12),each of which will be described later. The latching head 24B, whichserves to be latched together with latching pawls of the lid 31 of thenucleic acid amplification cartridge 3 to be described later, extendsradially outward.

The flange 2, which serves to be engaged with a stepped portion 27A ofthe housing vessel 27 when the nucleic acid extraction element 20 isplaced into the housing vessel 27 of the nucleic acid purificationcartridge 2 to be described later, is formed like a ring extendedradially outward (See FIG. 12).

The holding portion 26, which serves to hold a solid matrix 23, includesa tapered portion 26A, a pin-shaped portion 26B and a holding tab 26C.The tapered portion 26A helps cleansing liquid attached on the holdingportion 26 move downward. The pin-shaped portion 26B penetrates thesolid matrix 23. The holding tab 26C prevents the solid matrix 23 fromslipping off the pin-shaped portion 26B (holding portion 26) when thepin-shaped portion 26B penetrates the solid matrix 23.

Slightly above the holding portion 26 of the holding member 22, anO-ring 22A is fixed. As shown clearly in FIG. 13B, the O-ring 22Aassures a tight fit of the nucleic acid extraction element 20 to aninner surface of the reaction vessel 34 in the nucleic acidamplification cartridge 3 when the element is housed in the reactionvessel 34. When the nucleic acid extraction element 20 is housed in thereaction vessel 34, a sealed space is formed below the O-ring 22A whichmakes a tight contact with the inner surface of the reaction vessel 34.Since the O-ring 22A is above the holding portion 26, the solid matrix23 is housed in the sealed space.

The solid matrix 23, which serves to retain the nucleic acid in thesample, is provided by e.g. a filter paper impregnated with reagents forextracting the nucleic acid. The solid matrix 23 is formed in discalshape. The solidmatrix 23 is supported horizontally or substantiallyhorizontally, i.e. perpendicularly to a vertical axis of the holdingmember 22, and penetrated by the pin-shaped portion 26B.

Examples of the reagents usable for extracting nucleic acid include weakbases, chelate reagents, a combination of an anionic surfactant oranionic detergent and uric acid or a uric acid salt, and a combinationof a nucleic acid adsorbent carrier and an adsorption accelerator. Asthe nucleic acid adsorbent carrier, a variety of known materials may beemployed, and silica bead is typically used. The adsorption acceleratormay be provided by anything as long as it destroys cell membrane ordenaturalizes protein in the sample, and helps absorption of the nucleicacid onto the nucleic acid adsorbent carrier: Examples includeschaotropic materials (such as guanidine thiocyanate and other acid saltsof guanidine). The solid matrix 23 can be anything as long as it is ableto adsorb nucleic acid in the sample, and is not limited to thoseexemplified above.

According to the nucleic acid extraction element 20 described above, itis possible to keep the solid matrix 23 spaced from the bottom ofreaction vessel 34 when the nucleic acid extraction element 20 is housedin the reaction vessel 34 of the nucleic acid amplification cartridge 3described later. This prevents the solid matrix 23 from locating on aphotometric path for the photometry mechanism 8, improving accuracy inthe photometry. Since the solid matrix 23 is not on the photometricpath, it is possible to use the solid matrix 23 having a larger size.Therefore, it becomes possible to carry more nucleic acid in the solidmatrix 23, perform amplification of nucleic acid more efficiently, andimprove accuracy of the analysis.

As shown in FIG. 5 and FIG. 6, the cartridge main body 21 includes ahousing vessel 27, three cleansing vessels 28 ₁ through 28 ₃, a sampleholding vessel 29 and an surplus liquid removal vessel 21A, and is madeas a single piece by e.g. resin molding.

The housing vessel 27serves to house the nucleic acid extraction element20, and includes a stepped portion 27A to mate with the flange 25 of thenucleic acid extraction element 20. In order to prevent the nucleic acidextraction element 20 from getting out of an upper opening 27B of thehousing vessel 27 before the use of the nucleic acid purificationcartridge 2, it is preferable that the upper opening 27B is closed witha sealing member provided by e.g. a thin film of aluminum. The sealingmember may be configured to be peeled off by the user when the nucleicacid purification cartridge 2 is used, or to be peeled automatically inthe nucleic acid analyzer 1.

Each of the cleansing vessels 28 ₁ through 28 ₃ serves to hold cleansingliquid for removing impurities from the solid matrix 23 after loadingthe solid matrix 23 with nucleic acid. Preferably, the cleansing liquidis retained in the cleansing vessels 28 ₁ through 28 ₃ as part of thenucleic acid purification cartridge 2. Alternatively however, thecleansing liquid may be held in the nucleic acid analyzer 1 anddispensed into the cleansing vessels 28 ₁ through 28 ₃ at the time ofanalysis. As the cleansing liquid, material which seldom elutes thenucleic acid from the solid matrix 23 and prevents impurities frombonding can be used (Examples include guanidine hydrochloride andethanol). The three cleansing vessels 28 ₁ through 28 ₃may retain thesame cleansing liquid or may retain different cleansing liquids.

When cleansing liquid is preliminarily set into each of the cleansingvessels 28 ₁ through 28 ₃, upper openings 28A₁ through 28A₃ of thecleansing vessels 28 ₁ through 28 ₃ need to be sealed with sealingmembers such as thin film of aluminum. In this case, the upper openings28A₁ through 28A₃ of the cleansing vessels 28 ₁ through 28 ₃ may besealed individually, or all the three upper openings 28A₁ through 28A₃of the cleansing vessels 28 ₁ through 28 ₃ may be sealed together. Stillalternatively, all of the upper openings 28A₁ through 28A₃ of thecleansing vessels 28 ₁ through 28 ₃ and the upper opening 27B of thehousing vessel 27 may be sealed together.

The sample holding vessel 29 serves to hold a sample as an object ofanalysis (object from which nucleic acid is to be extracted). Holdingthe sample in the sample holding vessel 29 with the sample may be madebefore setting the nucleic acid purification cartridge 2 to the nucleicacid analyzer 1, or after setting the nucleic acid purificationcartridge 2 to the nucleic acid analyzer 1. In the latter case, it ispreferable that the nucleic acid analyzer 1 automatically dispenses thesample into the sample holding vessel 29. The sample may be provided bywhole blood, blood serum, blood plasma, urine, saliva or bodily fluid.

The surplus liquid removal vessel 21A serves to remove surplus cleansingliquid from the nucleic acid extraction element 20, the solid matrix 23and the holding portion 26 of the holding member 22 after the solidmatrix 23 in the nucleic acid extraction element is cleansed. Thesurplus liquid removal vessel 21A has a bottom wall 21Aa and a front andrear walls 21Ab, 21Ac on which water absorbing members 21Ad, 21Ae arefixed. The water absorbing members 21Ad, 21Ae are made of a porousmaterial such as resin foam and cloth, and is capable of absorbingsurplus cleansing liquid from the nucleic acid extraction element 20 bycontacting with the nucleic acid extraction element 20.

As shown in FIG. 8 and FIG. 9, the nucleic acid amplification cartridge3, which enables automatic amplification and measurement of nucleic acidin the nucleic acid analyzer 1, includes a cartridge main body 30 and alid 31.

The cartridge main body 30 includes four reagent vessels 32 ₁ through 32₄, a mixing vessel 33, and a reaction vessel 34, and is formed by e.g.resin molding, into a single piece.

Each of the reagent vessels 32 ₁ through 32 ₄ serves to hold a reagentor the like which is necessary for the amplification and measurement ofnucleic acid, in the form of an aqueous solution or a suspension. Thekind of the reagents to be held in each of the reagent vessels 32 ₁through 32 ₄ is selected in accordance with the amplification method andthe measurement method to be employed. Examples of the usableamplification method include PCR (Polymerase Chain Reaction) method,ICAN (Isothermal and Chimeric Primer-initiated Amplification of Nucleicacid) method, LAMP (Loop-Mediated Isothermal Amplification) method andNASBA (Nucleic acid Sequence Basea Amplification) method. Reagents usedin PCR method are at least two kinds of primers, a dNTP, and a DNApolymerase. Reagents used in ICAN method are a chimera primer, a DNApolymerase, and an RNaseH. Reagents used in LAMP method are one or morekinds of LAMP primers, adNTP, strand displacement DNA synthetase, and areverse transcriptase. Reagents used in NASBA method are at least twokinds of primers, a dNTP, an rNTP, a reverse transcriptase, aDNApolymerase, aRNaseH, and an RNA polymerase. On the other hand, usablemeasurement methods include fluorescence measurement, colorationmeasurement, radioactivity measurement or electrophoresis. It should benoted that the present description assumes that the nucleic acidanalyzer 1 uses fluorescence measurement method. In this case, use of afluorescent primer is preferred.

The mixing vessel 33 is used when two or more regents held in thereagent vessels 32 ₁ through 32 ₄ are mixed with each other beforesupplying to the reaction vessel 34.

It is preferable that the reagent vessels 32 ₁ through 32 ₄ preliminaryhold the reagents, but the reagents may be holded in the nucleic acidanalyzer 1 and dispensed later into the reagent vessels 32 ₄ through 32₄. In this case, the reagent vessels 32 ₁through 32 ₄must have theirupper openings 32A₁through 32A₄ be sealed with a sealing member providedby e.g. a thin film of aluminum. The upper openings 32hA₁ through 32A₄of the reagent vessels 32 ₁ through 32 ₄ may be sealed individually, orall the four upper openings 32A₁ through 32A₄ of the reagent vessels 32₁ through 32 ₄ may be sealed together. Still alternatively, all theupper openings 32A₁ through 32A₄ and 33A of the reagent vessels 32 ₁through 32 ₄ and of the mixing vessel 33 may be sealed together..

The reaction vessel 34 serves to hold a reagent mix and the nucleic acidextraction element 20, and provides a reaction space for reactionsbetween the nucleic acid supported by the nucleic acid extractionelement 20 and the reagent mix prepared in the mixing vessel 33 (SeeFIG. 13 and FIG. 14). The reaction vessel 34 has a cylindrical part 35and a reaction detection part 37.

The cylindrical part 35 to which the lid 31 is fitted has an innercircumferential wall formed with a thread groove 35A.

The reaction detection part 37 provides a space for amplificationreactions of nucleic acid, and functions as a detection vessel forfluorescence measurement. Specifically, the reaction detection part 37is a portion exposed to light from a light emitting part 80 of thephotometry mechanism 8 to be described later (See FIG. 15).

The lid 31, which determines whether the inside space of the reactiondetection part 37 is sealed or not, is attachable/detachable to and fromthe reaction vessel 34 (cylindrical part 35). More specifically, the lid31 is attached to the cylindrical part 35 or completely be detached fromthe cylindrical part 35 (reaction vessel 34) by turning. The lid 31includes a cylindrical main body part 38, a flange 39 and a holdingportion 36.

The main body part 38 has a thread ridge 38A to set to a thread groove35A of the cylindrical part 35 in the reaction vessel 34, and a recess38B for insertion of a turning member 60 (See FIG. 11B) in the lidattaching/removing mechanism 6 to be described later. The recess 38B hasan inner circumferential surface provided with a plurality of ribs 38C.The ribs 38C are spaced circumferentially at an interval, and extendvertically. Each rib 38C has a tapered upper end which becomes narrowertoward the tip.

The flange 39 allows engagement by pawls 64 of the outer-coat member 61in the lid attaching/removing mechanism 6 to be described later, whenthe lid removed from the reaction vessel 34 is moved (See FIG. 11B). Theflange 39 extends radially outward from an upper end of the main bodypart 38, like a ring.

As shown in FIG. 7B, the holding portion 36 serves to hold the nucleicacid extraction element 20 in the nucleic acid purification cartridge 2,and has a pair of latching pawls 36A, and a pin 36B.

The latching pawls 36A, which serve to engage with a latching head 24Bof the nucleic acid extraction element 20, protrude downward from abottom surface 38D of the main body part 38. Each latching pawl 36A hason its tip a hook portion 36Aa. The hook portion 36Aa moves pivotally.The hook portions 36Aa of the latching pawls 36A can move closer to andaway from each other.

The pin 36B is to be inserted into a recess 24A of the cylindrical part24 of the nucleic acid extraction element 20, and protrudes downwardfrom the bottom surface 38D of the main body part 38. The pin 36B servesas a guide when the nucleic acid extraction element 20 is held by thelid 31, and after the nucleic acid extraction element 20 is held by thelid 31, serves to reduce rattling of the nucleic acid extraction element20 with respect to the lid 31.

As shown in FIG. 1, the nucleic acid analyzer 1 includes a case 10provided with a lid 11, a display portion 12 and an operation portion13. The lid 11 determines whether the inside of the case 10 is exposedor not. The lid 11 is opened when the cartridges 2, 3 is placed on orremoved from the case 10 whereas the lid 11 is closed at the time ofnucleic acid analysis or when the apparatus is not in use. The displayportion 12 serves to display results of analysis etc., and is providedby an LCD, for example. The operation portion 13 serves to performvarious settings or initiating analysis or the like.

As shown in FIG. 2 and FIG. 3, the case 10 houses therein, a pipetteunit 4, a nucleic acid purifying operation mechanism 5, a lidattaching/removing mechanism 6, a temperature controlling mechanism 7,and a photometry mechanism 8 to be described later.

The pipette unit 4, which serves primarily for preparation of a mixtureliquid in the nucleic acid amplification cartridge 3, has a nozzle 40.The pipette unit 4 is used to supply a sample or cleansing liquid to thenucleic acid purification cartridge 2 as necessary.

The nozzle 40 is connected with an unillustrated pump for suckingin/discharging out a liquid, and switches a state in which a suckingforce is applied inside the nozzle 40 and a state in which a dischargingforce is applied inside. The nozzle 40 is movable in up-and-downdirections as well as horizontal directions by a drive mechanism such asa robot arm (not illustrated) controlled by a controller 10 whichincludes a CPU, for example. The nozzle 40 can be moved to the reagentvessels 32 ₁ through 32 ₄, the mixing vessel 33 and the reaction vessel34 in the nucleic acid amplification cartridge 3 as well as the housingvessel 27 in the nucleic acid purification cartridge 2. When the nozzle40 is used in preparation of a mixed sample or dispensing a mixed sampleinto the reaction vessel 34 (reaction detection part 37), a tip 43 isattached to a tip portion 42 as shown in FIG. 3. As shown in FIG. 2, thetips 43 are held in a rack 44 adjacent to a stand-by position for thenozzle 40 (pipette unit 4). Near the rack 44 is disposed a disposal box45 for disposal of used tips 43.

As shown in FIG. 2 through FIG. 4 and FIG. 10, the nucleic acidpurifying operation mechanism 5 serves to control operations of thenucleic acid extraction element 20 when nucleic acid in the sample isextracted by using the nucleic acid extraction element 20 of the nucleicacid purification cartridge 2. The nucleic acid purifying operationmechanism 5 includes a plurality of insertion pins 50, cylindricalbodies 51 and a support frame 52.

The insertion pins 50, each of which serves to fit into the cylindricalpart 24 of the nucleic acid extraction element 20, are supported by thesupport frame 52 movably therewith.

The cylindrical body 51 serves to remove the nucleic acid extractionelement 20 from the insertion pin 50 and covers the insertion pin 50 soas to be movable in up-and-down directions independently from theinsertion pin 50. Specifically, the cylindrical body 51 resides abovethe nucleic acid extraction element 20 (stand-by position) any timeexcept when removing the nucleic acid extraction element 20 from theinsertion pin 50, whereas it is moved relatively below the insertion pin50 when removing the nucleic acid extraction element 20 from theinsertion pin 50.

The support frame 52 supports the insertion pins 50 in a predeterminedinterval along the row of the nucleic acid purification cartridges 2,and serves as a carrier to move these insertion pins 50. The supportframe 52 is movable in up-and-down directions as well as fore-and-aftdirections by an unillustrated drive mechanism, whose operation iscontrolled by e.g. the controller 10 shown in FIG. 2. Therefore, theinsertion pins 50 and the nucleic acid extraction elements 20 attachedthereto can move in up-and-down directions and fore-and-aft directionswith the support frame 52. With this arrangement, a plurality of thenucleic acid extraction elements 20 can perform impregnation of a sampleto solid matrices 23, cleansing of the solid matrices 23 and removal ofsurplus solution in a simultaneous sequence (See FIG. 10).

As shown in FIG. 11 and FIG. 13, the lid attaching/removing mechanism 6serves to remove the lid 31 from the reaction vessel 34 of the nucleicacid amplification cartridge 3 or to attach the lid 31 to the reactionvessel 34, and includes a turning member 60 and an outer-coat member 61.The turning member 60 and the outer-coat member 61 are movable inup-and-down directions as well as horizontal directions by anunillustrated drive mechanism, and is controlled by the controller 10(See FIG. 2).

The turning member 60 serves to apply a rotating force to the lid 31 ofthe nucleic acid amplification cartridge 3, to hold the lid 31, and tomove the lid 31, and includes a generally columnar tip portion 62. Thetip portion 62 of the turning member 60 is formed with a plurality ofribs 63. The ribs 63 are spaced circumferentially of the tip portion 62at an interval, and extend vertically. Each of the ribs 63 has a taperedlower end which becomes narrower toward the tip. These ribs 63 engagewith the ribs 38C of the lid 31 as shown in FIG. 14, nesting alternatelywith adjacent ribs 38C in the recess 38B when the tip portion 62 isinserted.

According to the present configuration, when the tip portion 62 of theturning member 60 is rotated, the ribs 63 of the tip portion 61 and theribs 38C of the recess 38B interfere with each other, so it is possibleto prevent slipping rotation of the tip portion 62 in the recess 38B ofthe lid 31, and to apply the rotating force of the turning member 60appropriately to the lid 31. Further, the ribs 38C in the recess 38B aretapered so they become narrower toward the upper end whereas the ribs 63in the tip portion 61 in the turning member 60 are tapered so theybecome narrower toward the lower end. Therefore, it is possible toinsert the tip portion 61 of the turning member 60 into the recess 38Bof the lid 31 easily and reliably.

The outer-coat member 61 provides an outer sheath to the turning member60, and is cylindrical. The outer-coat member 61 has pawls 64 to engagewith the flange 39. Each pawl 64 has a tip portion 64 and a hook portion65 provided thereto, and the hook portion 65 can move pivotally. Thepawls 64 are engaged with the flange 39 of the lid 31 when the tipportion 62 of the turning member 60 is inserted into the recess 38B ofthe lid 31. This locks the turning member 60 to the lid 31 as a singlepiece, making possible to move the lid 31 by moving the turning member60 and the outer-coat member 61. The pawls 62 are automatically releasedfrom the engagement with the lid 31 when the turning member 60re-attaches the lid 31 to the reaction vessel 34.

As shown in FIG. 15, the temperature controlling mechanism 7 serves tocontrol the temperature of a heat block 70, thereby controlling thetemperature of a liquid held in the reaction detection part 37 in thenucleic acid amplification cartridge 3. The temperature of the heatblock 70 is monitored by an unillustrated temperature sensor, and inaccordance with results of the monitoring by the temperature sensor, afeedback control is provided to the heat block 70. The heat block 70 isformed with a recess 71 which is shaped correspondingly to an outershape of the reaction detection part 37 of the nucleic acidamplification cartridge 3. With is arrangement, it is possible toprovide selective and efficient temperature control to the reactionvessel 34 in the heat block 7. Further, the heat block 70 is providedwith a straight through holes 72, 73 connecting with the recess 71. Thethrough hole 72 provides a path for light which is emitted from a lightemitting part 80 of the photometry mechanism 8 to be described later, tocome to the reaction detection part 37 in the reaction vessel 34. Thethrough hole 73 provides a path for light which has passed the reactiondetection part 37 to come to a light receiving part 81.

The photometry mechanism 8 includes the light emitting part 80 and thelight receiving part 81. The light emitting part 80 sends an excitinglight to the reaction detection part 37 via the through hole 72. Thelight receiving part 81 receives fluorescent light when the excitinglight is thrown to the reaction detection part 37 via the through hole73. In the photometry mechanism 8, the light emitting part 80 emits theexciting light continuously while the light receiving part 81 monitorsthe amount of the fluorescent light continuously, whereby the extent ofthe amplification of nucleic acid is obtained at real-time.

Next, description will cover an operation in the nucleic acid analyzer1.

To perform an analysis of nucleic acid with the nucleic acid analyzer 1,firstly, nucleic acid purification cartridges 2 and nucleic acidamplification cartridges 3 are set to the nucleic acid analyzer 1 asshown in FIG. 1 through FIG. 4. The number of the cartridges 2, 3 to beset is discretionary as long as the number of nucleic acid purificationcartridges 2 and the number of nucleic acid amplification cartridges 3are equal to each other. The following description assumes that thenucleic acid purification cartridge 2 has its cleansing vessels 28 ₁through 28 ₃ containing cleansing liquid, and the sample holding vessel29 is already loaded with a sample before the nucleic acid purificationcartridge 2 is set to the nucleic acid analyzer 1.

Next, in accordance with the number of the cartridges 2, 3 and the kindsof the cartridges 2, 3 set in the nucleic acid analyzer 1, settings(purification method, amplification method and measurement method) aremade by using the operation portion 13 provided in the nucleic acidanalyzer 1 while making conformation on the display portion 12. Oncethese settings are complete, the nucleic acid analyzer 1 automaticallyperforms purification, amplification and measurement of nucleic acid.

As shown in FIG. 4, the purification of nucleic acid is performed in thenucleic acid purification cartridge 2 by moving the nucleic acidextraction element 20 by the nucleic acid purifying operation mechanism5.

More specifically, first, with the insertion pin 50 of the nucleic acidpurifying operation mechanism 5 positioned right above the housingvessel 27 of the container 21 of the nucleic acid purification cartridge2, the support frame 52 is driven to lower and then lift. By loweringthe insertion pin 50, the insertion pin 50 is fitted into thecylindrical part 24 of the nucleic acid extraction element 20, makingthe nucleic acid extraction element 20 locked to the nucleic acidpurifying operation mechanism 5. As the insertion pin 50 is lifted, thenucleic acid extraction element 20 is lifted by the nucleic acidpurifying operation mechanism 5.

Next, as shown in FIG. 10, the insertion pin 50 is moved together withthe support frame 52 to soak the solid matrix 23 of the nucleic acidextraction element 20 into a sample 29L which is held in the sampleholding vessel 29 of the nucleic acid purification cartridge 2, allowingthe solid matrix 23 to carry nucleic acid in the sample 29L.

Next, the solid matrix 23 is soaked sequentially into cleansing liquids28L₁ through 28L₃ held in the three cleansing vessels 28 ₁ through 283.More specifically, cleansing of the solid matrix 23 is performed bymoving the solid matrix 23 in up-and-down directions by the nucleic acidpurifying operation mechanism 5 in each cleansing vessel 28. In thisstep, the nucleic acid purifying operation mechanism 5 is controlled torepeat a cycle of a state in which the solid matrix 23 is completelysubmerged into the cleansing liquids 28L₁ through 28L₃ and a state inwhich the solid matrix 23 is above the surface of the cleansing liquids28L₁ through 28L₃.

In such a cleansing method as the above, the solid matrix 23 is slammedonto the liquid surface when moved from the state in which the solidmatrix 23 is above the liquid surface to the state in which it issubmerged into the cleansing liquids 28L₁ through 28L₃. In this moment,the solid matrix 23 comes under a big load since the solid matrix 23 isheld horizontally or substantially horizontally. On the other hand, whenthe solid matrix 23 is being moved in the cleansing liquids 28L₁ through28L₃, the solid matrix 23 comes under a big transfer resistance sincethe solid matrix 23 is held horizontally or substantially horizontally,providing a load which creates convection flow in the cleansing liquid.In these movement and actions, it is possible to remove impuritiesefficiently from the solid matrix 23. This effectively reducesinhibition caused by the impurities to the amplification of nucleic acidto be performed in the process of amplifying the nucleic acid, enablingto perform the analysis of the nucleic acid accurately. Such advantagesare obtained not only when the solid matrix 23 is moved horizontally butalso when the solid matrix 23 is moved while held at an angle withrespect to the vertical axis.

Finally, a tip portion of the nucleic acid extraction element 20 isbrought into contact with the water absorbing members 21Ad, 21Ae whichare held in the surplus liquid removal vessel 21A. The water absorbingmember 21Ad is disposed to make contact with the bottom wall 21Aa andthe front and the rear walls 21Ab, 21Ac of the surplus liquid removalvessel 21A. So if the tip portion of the nucleic acid extraction element20 is brought into contact with all portions of the water absorbingmember 21Ad, surplus cleansing liquid is removed efficiently from thetip portion of the nucleic acid extraction element 20, primarily fromthe solid matrix 23 and the holding portion 26 of the holding member 22.As a result, it becomes possible to reduce inhibition caused by theimpurities in the cleansing liquid to the amplification of the nucleicacid to be performed later by using the nucleic acid extraction element20.

It should be appreciated that after the completion of the cleansing, thesolid matrix 23 may be dried by air blow as held by the nucleic acidpurifying operation mechanism 5. After completing the cleansing (ordrying by air depending upon the case) of the solidmatrix 23, thenucleic acid extraction element 20 is detached from the insertion pin50, and the nucleic acid extraction element 20 is re-placed into thehousing vessel 27 in the nucleic acid purification cartridge 2. Removalof the nucleic acid extraction element 20 from the insertion pin 50 isperformed as described earlier, by lowering the cylindrical body 51 ofthe nucleic acid purifying operation mechanism 5 thereby causing thecylindrical body 51 to interfere with the latching head 24B.

As described, according to the nucleic acid purification cartridge 2,the solid matrix 23 is moved easily in the nucleic acid analyzer 1 byusing a solid (nucleic acid extraction element 20) which supports thenucleic acid. In this regards, the nucleic acid purification cartridge 2contributes to automated analysis of nucleic acid.

Amplification of nucleic acid is performed by preparing a reagent mix inthe nucleic acid amplification cartridge 3, dispensing the mixture intothe reaction vessel 34 in the nucleic acid amplification cartridge 3,and then placing the solidmatrix 23 which supports the nucleic acid intothe reaction vessel 34, together with the holding member 22. It shouldbe noted that if the reagent mix and the solid matrix 23 are heldtogether in the reaction vessel 34, temperature control is provided tothe heat block 70 (See FIG. 15) in accordance with the amplificationmethod employed, whereby temperature control is provided to the reactionvessel 34.

Preparation of the reagent mix is performed by first attaching a tip 43to the nozzle of the pipette unit 4, then dispensing predeterminedamounts of the reagents sequentially from the reagent vessels 32 ₁through 32 ₄ in the nucleic acid amplification cartridge 3 to the mixingvessel 33, and then mixing the dispensed liquids in a pipettingoperation by the pipette unit 4 (See FIG. 3).

Dispensing of the compound liquid to the reaction vessel 34 is performedby the pipette unit 4 after the lid attaching/removing mechanism 6removes the lid 31 from the reaction vessel 34. As shown in FIG. 11A andFIG. 11B, removal of the lid 31 by the lid attaching/removing mechanism6 is performed by inserting the tip portion 62 of the turning member 60of the lid attaching/removing mechanism 6 into the recess 38B of the lid31, and then lifting the turning member 60 while it is rotated. When theturning member 60 is inserted into the recess 38B, the hook portions 65of the pawls 64 in the outer-coat member 61 engage with the flange 39 ofthe lid 31. Therefore, it is possible to move the lid 31, which wasremoved from the reaction vessel 34, together with the turning member 60and the outer-coat member 61. In this way, the nucleic acid analyzerland the nucleic acid amplification cartridge 3 embody innovations foreasy and reliable removal of the lid 31 from the nucleic acidamplification cartridge 3 toward the goal of automated amplification ofnucleic acid and further, fully automated analysis of nucleic acid.

On the other hand, placing of the solid matrix 23 into the reactionvessel 34 is achieved by using the lid attaching/removing mechanism 6and the lid 31 of the nucleic acid amplification cartridge 3. Morespecifically, as shown in FIG. 12 and FIG. 13, placing of the solidmatrix 23 is accomplished by a series of operations such as holding ofnucleic acid extraction element 20 by the lid 31, and re-attaching thelid 31 to the reaction vessel 34.

As shown in FIG. 7B and FIG. 12, holding of the nucleic acid extractionelement 20 by the lid 31 is accomplished by first causing the lidattaching/removing mechanism 6 to move the lid 31 above the housingvessel 27in the nucleic acid purification cartridge 2, and then bylowering the lid 31. In the process of lowering the lid 31, the pin 36Bof the lid 31 is inserted into the recess 24A of the cylindrical part 24in the nucleic acid extraction element 20. This fixes the positionalrelationship of the lid 31 with respect to the cylindrical part 24 ofthe nucleic acid extraction element 20, and a pair of latching pawls 36Ain the lid 31 is brought appropriately in alignment with the latchinghead 24B of the cylindrical part 24. As the latching pawls 36A arepressed onto the latching head 24B from above, the latching pawls 36Aare displaced so that their hook portions 36Aa move away from eachother. As the latching pawls 36A are further lowered, the pin 36B of thelid 31 is inserted further into the recess 24A of the cylindrical part24, and as the hook portions 36Aa come below the latching head 24B, thehook portions 36Aa come closer to each other. As a result, the latchingpawls 36A make an engagement with the latching head 24B, capturing thenucleic acid extraction element 20 in the lid 31. This situation ishighly stable since the pin 36B of the lid 31 is inserted into therecess 24A of the cylindrical part 24, enabling to reduce rattles of thenucleic acid extraction element 20 in the lid 31.

As shown in FIG. 13, re-attaching of the lid 31 is achieved by bringingthe lid 31 into alignment with the reaction vessel 34, and then rotatingthe turning member 60 which has a hold on the lid 31. Specifically, byapplying a rotating force to the lid 31 which is in alignment with thetarget, the lid 31 is threaded into the cylindrical part 35 of thereaction vessel 34. Upon threading of the lid 31 into the cylindricalpart 35, the pawls 64 of the outer-coat member 61 cease their engagementwith the flange 39 of the lid 31, releasing the turning member 60 andthe outer-coat member 61 for movement independent from the lid 31. Onthe other hand, since the lid 31 holds the nucleic acid extractionelement 20, the nucleic acid extraction element 20 sits in the reactionvessel 34. Since the nucleic acid extraction element 20 has the O-ring22A at a position slightly above the holding portion 26 as describedearlier, the solid matrix 23 of the nucleic acid extraction element 20is fixed in the sealed space, at a predetermined distance above thebottom of the reaction vessel 34. The reaction detection part 37 alreadyhas a reagent mix, and the solid matrix 23 is entirely soaked therein inthe reaction detection part 37, so the nucleic acid elutes from thesolid matrix 23 and the eluted nucleic acid reacts with the reagents toamplify.

As described, according to the nucleic acid analyzer 1, it is possibleto move the nucleic acid extraction element 20 from the housing vessel27 to the reaction vessel 34 and to set the element therein by using thelid attaching/removing mechanism 6 which is a mechanism needed forattaching/detaching the lid 31. Specifically, according to the nucleicacid analyzer 1, there is no need for a specific mechanism for movingthe nucleic acid extraction element 20. This enables to avoid complexityof the apparatus, and thereby enabling to reduce size increase of theapparatus when performing both purification of nucleic acid andamplification of the nucleic acid within a single piece of equipment. Asanother advantage, this enables to reduce increase in the number ofoperation mechanisms which need to be controlled in the apparatus.

As shown in FIG. 15, measurement of the nucleic acid is performed by thephotometry mechanism 8 while the reaction vessel 34 is covered by alight shielding member 9 from above.

In the photometry mechanism 8, an exciting light is thrown from thelight emitting part 80 to the reaction detection part 37 of the reactionvessel 34 while a fluorescent light generated in the reaction detectionpart 37 is received by the light receiving part 81. As describedearlier, the solid matrix 23 is placed at a position not interruptingthe photometric measurement by the photometry mechanism 8, so accuratemeasurement of nucleic acid is possible according to the nucleic acidanalyzer 1.

As has been described thus far, according to the nucleic acid analyzer1, it is possible to perform automated analysis of nucleic acid simplyby only setting the nucleic acid purification cartridges 2 and thenucleic acid amplification cartridges 3 of the above-describedconfiguration. The nucleic acid purification cartridge 2 and the nucleicacid amplification cartridge 3 also embody a number of innovations whichenables the automated analysis of nucleic acid. Therefore, when thenucleic acid analyzer 1, the nucleic acid purification cartridge 2 andthe nucleic acid amplification cartridge 3 are used, there is noprocedure which must depend upon manual operation by the user other thansetting the cartridges 2, 3 into the nucleic acid analyzer 1, throughoutthe entire operation of extracting nucleic acid and amplifying thenucleic acid. Therefore, remarkable reduction is achieved in the burdenon the user in the nucleic acid analysis. Further, there is now noconcern for different levels of skill among the users which may lead topoor repeatability due to inconsistency in the recovery rate of nucleicacid.

The present invention is not limited to the examples covered by theembodiment described above. The solid matrix, for example, of thenucleic acid extraction element may not necessarily be held horizontallyor substantially horizontally with respect to the vertical axis of theholding member. The solid matrix may not necessarily be formed discal,and the way the holding member holds the solid matrix is not limited topenetration through the solid matrix. Further, the lid of the nucleicacid amplification cartridge may not necessarily be totally separablefrom the cartridge main body, and may open and close the upper openingof the reaction vessel as a non-detachable member of the cartridge mainbody.

Next, description will be made for a second embodiment of the presentinvention, with reference to FIG. 16 through FIG. 33. Note however, thatin the drawings referred to hereinafter, elements which are identicalwith or similar to those described in the first embodiment are indicatedby the same reference codes, and description therefore will not berepeated.

FIG. 16 through FIG. 18 show a nucleic acid analyzer 1′. Like thenucleic acid analyzer 1 (See FIG. 1 and others) which is alreadycovered, when using this analyzer, a plurality of nucleic acidpurification cartridges 2′ and the same number of nucleic acidamplification cartridges 3′ are set. As shown in FIG. 17, the apparatusincludes a pipette unit 4′ and a nucleic acid purifying operationmechanism 5′.

As shown in FIG. 19, the nucleic acid purification cartridge 2′ is acomponent that enables automatic purification of nucleic acid in thenucleic acid analyzer 1′, and includes a nucleic acid extraction element20′ and a cartridge main body 21′.

The nucleic acid extraction element 20′ serves to support nucleic acidin the sample, and includes a holding member 22′ and a solid matrix 23′as shown clearly in FIG. 20A through FIG. 20C. The holding member 22′has a cylindrical part 24′, a flange 25′, and a clip portion 26′, and isformed entirely by resin molding for example.

The cylindrical part 24′ serves to move the nucleic acid extractionelement 20′ (See FIG. 18 and FIG. 22), and has a recess 24A′, cutouts24B′, 24C′, and a plurality of ribs 24D′. The recess 24A′ serves toaccept a tip portion 42′ (See FIG. 26A and FIG. 26B) of a nozzle 40′ inthe pipette unit 4′ to be described later or an insertion pin 50′ of anucleic acid purification mechanism 5′ (See FIG. 18), and is formed in acolumnar shape. The cutouts 24B′, 24C′ serve to give elasticity to thecylindrical part 24′, and include a pair of V-shaped cutouts 24B′ and arectangular through hole 24C′. Specifically, when the recess 24A′ isfitted by the tip portion 42′ of the nozzle 40′ or the insertion pin 50′(See FIG. 18 and FIG. 22), the cutouts 24B′, 24C′ exert an elastic forceonto these members thereby ensuring the fit. When the recess 24A′ isfitted by the tip portion 42′ of the nozzle 40′ or the insertion pin50′, the ribs 24D′ exert a friction force onto these members therebyensuring the fit. The ribs extend in up-and-down directions in an innersurface of the cylindrical part 24′.

The flange 25′ is formed in the shape of a ring extended radiallyoutward. The flange 25′ serves to sit into a stepped portions 27A′, 36′provided at a target location when holding the nucleic acid extractionelement 20′ at the target location (a housing vessel 27 of the nucleicacid purification cartridge 2′ and a reaction vessel 34′ of the nucleicacid amplification cartridge 3′) (See FIG. 21 and FIG. 33).

The clip portion 26′, which clips an end of a solid matrix 23′ tocombine the solid matrix 23′ with the holding member 22′, is provided bya pair of clip blades 26 a′. The clip blades 26 a′ should preferably bemade to-have as small area of contact with the solid matrix 23′ aspossible for increased recovery rate of the nucleic acid. The reason forthis is, as will be described later, the nucleic acid is allowed toelute and is collected after allowing the solid matrix 23′ to supportnucleic acid, but it is not easy to elute the nucleic acid from aportion of the solid matrix 23′ pinched by the clip blades 26 a′.

The solid matrix 23′, which serves as a carrier of nucleic acid in thesample, is provided by e.g. a filter paper impregnated with reagents forextracting the nucleic acid. The solid matrix 23′ is formed in the shapeof a strip, having its end clipped by the clip portion 26′ therebysuspended below the holding member 22′.

As shown in FIG. 19 and FIG. 21, the cartridge main body 21′ includes ahousing vessel 27, three cleansing vessels 28 ₁ through 28 ₃, and asample holding vessel 29 similarly to the cartridge main body 21 (SeeFIG. 5 and FIG. 6) of the nucleic acid purification cartridge 2 which isdescribed earlier, but does not include an surplus liquid removal vessel21A (See FIG. 5 and FIG. 6). Obviously, the cartridge main body 21′ maybe provided with an surplus liquid removal vessel.

As shown in FIG. 23, FIG. 24A and FIG. 24B, the nucleic acidamplification cartridge 3′, which enables automatic amplification andmeasurement of nucleic acid in the nucleic acid analyzer 1′, includes acartridge main body 30′ and a lid 31′.

The cartridge main body 30′ has five reagent vessels 32′, a mixingvessel 33′, and a reaction vessel 34′, and these vessels 32′, 33′ and34′ are formed by e.g. resin molding, in a single piece.

Each reagent vessel 32 serves to hold a reagent or the like which isnecessary for the amplification and measurement of nucleic acid, in theform of an aqueous solution or a suspension.. Each reagent vessel 32′has a generally rectangular cross section. More accurately, four sidesurfaces 32A′ have their center region protruded inward. Therefore, thereagent vessel 32′ has its four corners acutely angled to be smallerthan 90 degrees. This reduces a tendency that the reagent stay attachedon the side walls 32′ of the reagent vessel 32′, enabling to keep thereagent to stay in the bottom region of the reagent vessel 32′. As aresult, it becomes possible to make efficient use of the reagent held inthe reagent vessel 32′. For example, when a very expensive reagent hasto be used, the amount of the reagent dispensed in the reagent vessel32′ may be small, which enables to reduce production cost. Such anadvantage is also obtained by providing grooves or ribs on the sidesurfaces 32A′ of the reagent vessel 32′.

With the above, the kind of the reagents to be held by each of thereagent vessels 32′ is selected in accordance with the amplificationmethod and the measurement methods to be employed. Examples of theusable amplification method include PCR method, ICAN method, LAMP methodand NASBA method.

The mixing vessel 33′ is used when two or more regents held in thereagent vessels 32′ are mixed with each other before supplying to thereaction vessel 34′. Like the reagent vessel 32′ which was describedabove, the mixing vessel 33′ also has acutely angled four cornerssmaller than 90 degrees. Obviously, side surfaces 33A′ of the mixingvessel 33′ may be provided with grooves or ribs.

The reaction vessel 34′ serves to hold a reagent mix and the nucleicacid extraction element 20, and provides a reaction space for reactionsbetween the nucleic acid supported by the nucleic acid extractionelement 20′ and the reagent mix prepared in the mixing vessel 33′ (SeeFIG. 33). The reaction vessel 34′ has a cylindrical part 35, a reactiondetection part 37, and a stepped portion 36′ provided therebetween. Thestepped portion 36′ serves as a seat for the flange 25′ of the nucleicacid extraction element 20′ (See FIG. 33), and is provided by reducingthe diameter of the reaction detection part 37 to be smaller than thediameter of the cylindrical part 35.

The lid 31′, which determines whether the inside space of the reactiondetection part 37 is sealed or not, is attachable/detachable to and fromthe reaction vessel 34′ (cylindrical part 35). More specifically, thelid 31′ can be turned to thread into the cylindrical part 35 (reactionvessel 34) or completely detached from the cylindrical part 35. Like thelid 31 of the nucleic acid amplification cartridge 3 described earlier(See FIG. 9), the lid 31′ includes a cylindrical main body part 38 and aflange 39. However, in the nucleic acid analyzer 1′, the nucleic acidextraction element 20′ is moved by using a nozzle 40′ of a pipette unit4′, and for this reason, lid 31′ does not include the holding portion 36(See FIG. 7B and FIG. 9) which is included in the lid 31 of the nucleicacid amplification cartridge 3 described earlier.

FIG. 16 and FIG. 17 show a pipette unit 4′, which serves primarily toprepare a compound liquid in the nucleic acid amplification cartridge 3′and to move the compound liquid to the reaction vessel 34′. As shown inFIG. 25 through FIG. 28, the pipette unit 4′ includes a nozzle 40′ and aremoval member 41′.

The nozzle 40′ is capable of sucking/discharging a liquid and movable inup-and-down directions and horizontal directions to reach the reagentvessels 32′, the mixing vessel 33′ the reaction vessel 34′ in thenucleic acid amplification cartridge 3′, and the housing vessel 27 inthe nucleic acid purification cartridge 2′ (See FIG. 16 and FIG. 17).When the nozzle 40′ is used in preparation of a mixed sample ordispensing the mixed sample into the reaction vessel 34′ (reactiondetection part 37), a tip 43 is attached to a tip portion 42′, as shownin FIG. 25A and FIG. 25B. In the nozzle 40′, an O-ring 42 a′ is fittedto a location on the tip portion 42′ where the tip 43 is attached, forincreased sealing between the tip portion 42′ and the tip 43 when thetip portion 42′ is fitted with the tip 43.

As shown in FIG. 22, the pipette unit 4′ also serves to take the nucleicacid extraction element 20′ out of the housing vessel 27of the nucleicacid purification cartridge 2′, and to move the nucleic acid extractionelement 20′ to the reaction vessel 34′ of the nucleic acid amplificationcartridge 3′ as shown in FIG. 31. For such an operation, the nucleicacid extraction element 20′ is attached to the tip portion 42′ of thenozzle 40′ as shown in FIG. 26A and FIG. 26B.

As shown in FIG. 27 and FIG. 28, the removal member 41′ serves to removethe tip 43 or the nucleic acid extraction element 20′ from the tipportion 42′ of the nozzle 40′. The removal member 41′ sheathes thenozzle 40′ and is movable in up-and-down directions independently fromthe nozzle 40′. Specifically, the removal member 41′ resides above anend surface 43 a of the tip 43 or the flange 25′ of the nucleic acidextraction element 20′ (stand-by position) any time except when removingthe tip 43 or the nucleic acid extraction element 2′ from the tipportion 42′ of the nozzle 40′, whereas it is moved down relatively belowthe nozzle 40′ when removing them. As the removal member 41′ is moved inthe downward direction from the stand-by position beyond a predetermineddistance, an end surface 41A′ of the removal member 41′ interferes withan end surface 43 a of the tip 43 or the flange 25′ of the nucleic acidextraction element 20′, exerting a downward force onto the tip 43 or thenucleic acid extraction element 20′, removing the tip 43 or the nucleicacid extraction element 20′ from the tip portion 42′ of the nozzle 40′.

As shown in FIG. 16 through FIG. 18, the nucleic acid purifyingoperation mechanism 5′ serves to control operation of the nucleic acidextraction element 20′ when nucleic acid in the sample is extracted byusing the nucleic acid extraction element 20′. Like the nucleic acidpurifying operation mechanism 5 of the nucleic acid analyzer 1 describedearlier (See FIG. 2 through FIG. 4), the nucleic acid purifyingoperation mechanism 5′ includes a plurality of insertion pins 50′,cylindrical bodies 51 and a support frame 52. However, each insertionpin 50′ is shaped like the tip portion 42′ of the nozzle 40′ forappropriate fitting into the cylindrical part 24′ of the nucleic acidextraction element 20′.

Next, description will cover an operation in the nucleic acid analyzer1′.

The nucleic acid analyzer 1′ automatically performs purification,amplification and measurement once nucleic acid purification cartridges2′ and nucleic acid amplification cartridges 3′ are set to the nucleicacid analyzer 1′ as shown in FIG. 16 through FIG. 18 and settings aremade for the number and the type of cartridges 2′, 3′ (purificationmethod, amplification method and measurement method).

As shown in FIG. 18, purification of nucleic acid is performed in thenucleic acid purification cartridge 2′ by moving the nucleic acidextraction element 20′ using the nucleic acid purifying operationmechanism 5′. More specifically, first, the insertion pins 50′ of thenucleic acid purifying operation mechanism 5′ are fitted intocorresponding cylindrical parts 24′ of the nucleic acid extractionelements 20′ so that the plurality of nucleic acid extraction elements20′ can be moved all together. Under this condition, the nucleic acidpurifying operation mechanism 5′ soaks the solid matrices 23′ of thenucleic acid extraction elements 20′ into samples, allowing each solidmatrix 23′ to be impregnated with nucleic acid in the sample.

Finally, the solid matrices 23′ are soaked sequentially into cleansingliquids in the three cleansing vessels 28 ₁ through 28 ₃ (See FIG. 19).More specifically, cleansing of the solid matrices 23′ is performed bymoving the solid matrices 23′ in up-and-down directions repeatedly ineach of the cleansing vessels 28 ₁ through 28 ₃ (See FIG. 19) by thenucleic acid purifying operation mechanism 5′. In this step, the nucleicacid purifying operation mechanism 5′ is controlled to repeat a cycle ofa state in which the solid matrices 23′ are completely submerged intothe cleansing liquid and a state in which the solid matrices 23′ areabove the surface of the cleansing liquids. According to the cleansingmethod as this, it is possible to remove impurities efficiently from thesolid matrices 23. This enables to effectively reduce inhibition causedby the impurities to the amplification of nucleic acid to be performedin the amplification of nucleic acid, and thereby to perform theanalysis of the nucleic acid accurately.

It should be appreciated that after the completion of the cleansing, thesolid matrices 23′ may be dried by air blow as held by the nucleic acidpurifying operation mechanism 5′. After completing the cleansing (ordrying by air depending upon the case) of the solid matrices 23 ′, thenucleic acid extraction elements 20′ are detached from the insertionpins 50 ′, and the nucleic acid extraction elements 20 are re-placedinto the housing vessels 27 in the nucleic acid purification cartridge2′ (See FIG. 19 and FIG. 21).

As described, according to the nucleic acid purification cartridge 2′,the target nucleic acid is moved easily in the nucleic acid analyzer 1′by using a solid (nucleic acid extraction element 20′) which supportsthe target nucleic acid. In this regards, the nucleic acid purificationcartridge 2′ contributes to automated analysis of nucleic acid.

Amplification of nucleic acid is performed by preparing a reagent mix inthe nucleic acid amplification cartridge 3′, dispensing the mixture intothe reaction vessel 34′ in the nucleic acid amplification cartridge 3′,and then moving the solid matrix 23′ which supports the nucleic acidinto the reaction vessel 34′ together with the holding member 22′. Itshould be noted that if the reagent mix and the solidmatrix 23′ are heldtogether in the reaction vessel 34′ as shown in FIG. 33, temperaturecontrol is provided to the heat block 70 in accordance with theamplification method employed, whereby temperature control is providedto the reaction vessel 34′.

Preparing of the reagent mix and dispensing of the reagent mix to thereaction vessel 34′ are performed by the pipette unit 4′ like in thenucleic acid analyzer 1 described earlier (See FIG. 1 and so on). Notethat before dispensing the reagent mix to the reaction vessel 34′, thelid 31′ must be removed from the reaction vessel 34′ by the lidattaching/removing mechanism 6. Such a removal of the lid 31′ isperformed as shown in FIG. 29 and FIG. 30, by inserting the turningmember 60 of the lid attaching/removing mechanism 6 into the recess 38B′of the lid 31′, and then lifting the turning member 60 while it isrotated. When the turning member 60 is inserted into the recess 38B′,the hooks 62 of the turning member 60 engage with the flange 39′ of thelid 31′, and therefore, it is possible to move the lid 31′ which wasremoved from the reaction vessel 34′ together with the turning member60. As exemplified, the nucleic acid analyzer 1′ and the nucleic acidamplification cartridge 3′ embody innovations for easy and reliableremoval of the lid 31′ from the nucleic acid amplification cartridge 3′toward the goal of automated amplification of nucleic acid and further,fully automated analysis of the nucleic acid.

On the other hand, moving of the solid matrix 23′ into the reactionvessel 34′ is accomplished by a series of operations such as removing ofthe nucleic acid extraction element 20′ from the housing vessel 27 ofthe nucleic acid purification cartridge 2′ (See FIG. 22), moving of thenucleic acid extraction element 20′ to the reaction vessel 34′ in thenucleic acid amplification cartridge 3′ and removing of the nucleic acidextraction element 20′ from the nozzle 40′ (See FIG. 28 and FIG. 31).

As shown in FIG. 22, the removal of the nucleic acid extraction element20′ is accomplished by first moving the nozzle 40′ right above thehousing vessel 27 in the nucleic acid purification cartridge 2′, andthen by lowering the nozzle 40′ to engage the tip portion 42′ of thenozzle 40′ with the cylindrical part 24′ of the nucleic acid extractionelement 20′, and then lifting the nozzle 40′. In this step, note thatthe cylindrical part 24′ is formed with the cutouts 24B′, 24C′ such asthe V-shaped cutouts 24B′ and the rectangular through hole 24C′ (SeeFIG. 20A through FIG. 20C). Therefore, when the tip portion 42′ of thenozzle 40′ is fitted into the cylindrical part 24′, it is possible toapply appropriate elastic force to the tip portion 42′. This ensuresappropriate holding of the nucleic acid extraction element 20′ in thecylindrical part 24′, with respect to the tip portion 42′ of the nozzle40′.

The movement of the nucleic acid extraction element 20′ is made bymoving the nozzle 40′ while the nucleic acid extraction element 20′ isheld by the tip portion 42′ of the nozzle 40′.

As shown in FIG. 28 and FIG. 31, the removal of the nucleic acidextraction element 20′ is accomplished by first moving the tip portion42′ of the nozzle 40′ to inside the reaction vessel 34′ together withthe nucleic acid extraction element 20′, and then lowering the removalmember 41′ relatively below the nozzle 40′. Specifically, when theremoval member 41′ is moved downward, the removal member 41′ interfereswith the flange 25′ of the nucleic acid extraction element 20′, applyinga downward force to the flange 25′ i.e. to the nucleic acid extractionelement 20′ to remove the nucleic acid extraction element 20′ from thetip portion 42′ of the nozzle 40′.

As described, according to the nucleic acid analyzer 1′, it is possibleto move the nucleic acid extraction element 20′ by using the nozzle 40′and the removal member 41′ which is a mechanism needed for preparationof samples. Since one of the components (pipette unit 4) which isessential for performing purification of nucleic acid and amplificationof the nucleic acid within a single piece of equipment serves for amultiple of tasks, complexity of the apparatus is avoided. Also, thisenables to reduce increase in the number of operation mechanisms whichneed to be controlled in the apparatus, offering another advantage inreducing complexity and size increase of the apparatus.

As shown in FIG. 31, the nucleic acid extraction element 20′, which isremoved from the tip portion 42′ of the nozzle 40′, is seated on itsflange 25′ of the holding member 22′ onto the stepped portion 36′ of thereaction vessel 34′. In this step, the solid matrix 23′ placed in thereaction detection part 37 has its lower end above the bottom of thereaction detection part 37 by a predetermined distance. The reactiondetection part 37 already has the reagent mix in it, so the solid matrix23′ is entirely soaked in the reaction detection part 37, wherebynucleic acid elutes from the solid matrix 23′ and the eluted nucleicacid reacts with the reagents and is amplified.

As described, the lower end of the solid matrix 23′ is above the bottomof the reaction detection part 37. More specifically, the solid matrix23′ has its lower end placed at a position not interrupting the travelof the exciting light to the reaction detection part nor interferingwith the measurement of the fluorescent light by the photometrymechanism 8 (See FIG. 33). Therefore, even if the nucleic acid issupported by a solid support, the solid support does not prevent thenucleic acid measurement.

As shown in FIG. 33, the measurement of the nucleic acid is performedafter re-attaching the lid 31′ of the reaction vessel 34′, by thephotometry mechanism 8 while the reaction vessel 34′ is covered by alight shielding member 9 from above. The photometry mechanism 8 performsessentially the same operation as described earlier for the nucleic acidanalyzer 1 (See FIG. 1 and so on) when measuring the nucleic acid.

As has been described thus far, according to the nucleic acid analyzer1′, again, it is possible to perform automated analysis of nucleic acidsimply by setting the nucleic acid purification cartridge 2′ and thenucleic acid amplification cartridge 3′ of the above-describedconfiguration, just like in the nucleic acid analyzer 1 (See FIG. 1,etc.) Therefore, there is no portion which must depend upon manualoperation by the user other than setting the cartridges 2′, 3′ to thenucleic acid analyzer 1′, throughout the entire operation of extractingnucleic acid and amplifying the nucleic acid. Therefore, remarkablereduction is achieved in the burden on the user in the nucleic acidanalysis, and further, there is now no concern for different levels ofskill among the users which may cause poor repeatability due toinconsistency in the recovery rate of nucleic acid.

EXAMPLES

In the following examples, a nucleic acid purification cartridge, anucleic acid amplification cartridge and a nucleic acid analyzeraccording to the first embodiment were employed to test if a targetnucleic acid provided by a human genome DNA is appropriately purifiedand amplified. The test method used was SNP (Single NucleotidePolymorphism) typing.

Example 1

(Formation of Nucleic Acid Purification Cartridge)

The nucleic acid purification cartridge was formed in the followingsteps: The cartridge main body (reference code 21 in the drawings) andthe nucleic acid extraction element (reference code 20 in the drawings)were formed in the method described in the next paragraph; then, thenucleic acid extraction element was placed in the housing vessel of thecartridge main body (reference code 27 in the drawings), and waterabsorbing members (reference codes 21Ad, 21Ae in the drawings) providedby a resin foam (Urethane Foam SAQ, manufactured by INOAC CORPORATION)were fixed in the surplus liquid removal vessel (reference code 21A inthe drawings). The size of the water absorbing member 21Ad was 5 mm×8mm×17 mm, while the size of the water absorbing members 21Ae was 5 mm×11mm×14 mm.

The cartridge main body was formed of PET, using resin injectionmolding, into a shape shown in FIG. 5 and FIG. 6.

The nucleic acid extraction element was formed by allowing the holdingmember (reference code 22 in the drawings) to hold a solid matrix(reference code 23 in the drawings) The solid matrix was formed bypunching FTA Classic Card (Whatman plc, Cat. No. WB120205) into a dischaving a 2.5 mm diameter. FTA Classic Card is a nucleic acid collectionfilter paper made primarily of cellulose. The holding member was formedof a PET by resin injection molding into the shape in FIG. 7A and FIG.7B. Note, however, that upon the resin injection molding, the holdingmember was not formed with the holding tab (reference code 26C in thedrawings). The holding tab was made by first creating a hole in thecenter of the solid matrix, inserting the matrix through the pin-shapedportion (reference code 26B in the drawings) of the holding member, andthen thermally deforming the tip portion of the pin-shaped portion. Asdescribed earlier, the holding tab prevents the solid matrix fromslipping out of the pin-shaped portion.

(Formation of Nucleic Acid Amplification Cartridge)

As for the nucleic acid purification cartridge, the cartridge main body(reference code 30 in the drawings) and the lid (reference code 31 inthe drawings) were formed of a PET by resin injection molding into theshapes shown in FIG. 8 and FIG. 9. After that, the lid was threaded intothe cartridge main body reaction vessel (reference code 34 in thedrawings)

(Purification of Nucleic Acid)

Purification of the nucleic acid was performed automatically by thenucleic acid analyzer, after dispensing a sample (reference code 29L inthe drawings) into the sample holding vessel (reference code 29 in thedrawings) of the nucleic acid purification cartridge main body,dispensing a cleansing liquid (reference code 28L₁ through 28L₃ in thedrawings) to each of three cleansing vessels (reference code 28 ₁through 28 ₃), and then setting the nucleic acid purification cartridgeinto the nucleic acid analyzer (reference code 1 in the drawings).

The sample was provided by whole blood (mixed with an anticoagulantHeparin Na), and the amount pipetted was 120 μL. The cleansing liquid28L₁ was provided by Cleansing liquid I (800 μL) in the following table,the cleansing liquid 28L₂ was provided by Cleansing liquid I (600 μL) inthe following table and the cleansing liquid 28L₃ was provided byCleansing liquid II (600 μL) in the following table. TABLE 1 CompositionpH Cleansing 10 mM   1 mM 8.0 Liquid I Tris-HCl EDTA Cleansing 10 mM 0.1mM 8.0 Liquid II Tris-HCl EDTA

In the nucleic acid analyzer, the nucleic acid purifying operationmechanism (reference code 5 in the drawings) was driven so that thenucleic acid extraction element (solid matrix) would follow an operationdescribed below:

First, the insertion pin (reference code 50 in the drawings) of thenucleic acid purifying operation mechanism was inserted into thecylindrical part (reference code 24 in the drawings) of the holdingmember, and the solid matrix was soaked into the whole blood in thesample holding vessel. Next, the solid matrix was cleansed in the threecleansing vessels 28 ₁ through 28 ₃. The cleansing of the solid matrixwas performed by sequentially using the cleansing vessels 28 ₁ through28 ₃ in the order of: cleansing vessel 28 ₁ cleansing vessel 28 ₂cleansing vessel 28 ₃. The cleansing of the solid matrix in thecleansing vessel 28 ₁ was performed by repeating a cycle of lifting thesolidmatrix 23 entirely above the liquid surface of the cleansing liquid28L₁ and lowering it entirely below the surface of the liquid 28L₁ for aperiod of one minute at a frequency of 20 Hz. On the other hand, thecleansing cycles for the solid matrix in the cleansing vessels 28 ₂, 28₃ were performed for a period of two minutes, with all the otheroperating conditions being the same as in the cleansing vessel 28 ₁.

Next, extra components which can inhibit the nucleic acid amplificationreaction to take place in the next step were removed. The removal of theextra components was performed by pressing the solid matrix and a tippart of the holding portion (the holding tab, the pin-shaped portion andtapered portion (reference code 26 in the drawings)) onto the waterabsorbing members (reference code 21Ad, 21Ae in the drawings).

(Verification of Nucleic Acid Amplification)

The amplification of the nucleic acid was verified by PCR method usingtwo reagent compound liquids A, B listed in the following Table 2. Thedegree of amplification of the nucleic acid was verified by SNP (SingleNucleotide Polymorphism) typing of the base sequence CYP2C19*2*3 whichis a code for drug-metabolizing enzyme. TABLE 2 40 μL Reagent Mix ASterile Distilled Water 35.6 μL 10× Gene Taq Universal Buffer (Mg free)4 μL (Manufactured by: Nippon Gene Co., Ltd.) 5 units/μl Gene Taq FP 0.4μL Reagent Mix B Sterile Distilled Water 5.6 μL 10× Gene Taq UniversalBuffer (Mg free) 4 μL (Manufactured by: Nippon Gene Co., Ltd.) 40%Glycerol liquid 20 μL 100 mM MgCl₂ liquid (Mfg: Nippon Gene Co., Ltd.)1.2 μL 2.5 mM dNTP Mixture (Mfg: Nippon Gene Co., Ltd.) 6.4 μL 100 μMCYP2C19*2 F-Primer (Sequence Number 1) 0.4 μL 100 μM CYP2C19*2 R-Primer(Sequence Number 2) 0.2 μL 100 μM CYP2C19*3 F-Primer (Sequence Number 3)0.2 μL 100 μM CYP2C19*3 R-Primer (Sequence Number 4) 0.4 μL 5 μMCYP2C19*2 probe (Sequence Number 5) 0.8 μL 5 μM CYP2C19*3 probe(Sequence Number 6) 0.8 μLSequence Number 1: gttttctcttagatatgcaataattttcccaSequence Number 2: cgagggttgttgatgtccatcSequence Number 3: gaaaaattgaatgaaaacatcaggattgtaSequence Number 4: gtacttcagggcttggtcaataSequence Number 5: ttatgggttcccgggaaataatc-(BODIPY-FL)Sequence Number 6: gcaccccctggatcc-(TAMRA)

More specifically, the verification of the nucleic acid amplificationwas performed automatically in the nucleic acid analyzer (reference code1 in the drawings), after pipetting the reagent compound liquid A or thereagent compound liquid B individually to the reagent vessel (referencecode 32 ₁, 32 ₂ in the drawings) of the nucleic acid amplificationcartridge main body, and then setting the nucleic acid amplificationcartridge in the nucleic acid analyzer.

In the nucleic acid analyzer, the pipette unit (reference code 4), thelid attaching/removing mechanism (reference code 6 in the drawings) andthe temperature controlling mechanism (reference code 7) were driven sothat the nucleic acid extraction element (solid matrix) would follow anoperation described below:

First, after the tip (reference code 43) was attached to the nozzle(reference code 40) of the pipette unit, the mixing vessel (referencecode 33 in the drawings) was loaded with 30 μL of the reagent compoundliquid A from the reagent vessel 33A and 30 μL of the reagent compoundliquid B from the reagent vessel 33B. Next, using a siphon operation ofthe nozzle, the reagent compound liquids A, B were agitated to mix witheach other for use as a reaction liquid. Then, using the nozzle, 50 μLof the reaction liquid was dispensed to the reaction vessel (referencecode 34 in the drawings).

Meanwhile, the turning member (reference code 60 in the drawings) of thelid attaching/removing mechanism removed the lid (31 in the drawings)from the nucleic acid amplification cartridge. The lid was moved tobring its latching pawls (reference code 36A in the drawings) inengagement with the engagement head (reference code 24B in the drawings)of the nucleic acid extraction element, to connect the two componentstogether.

Next, while the lid attaching/removing mechanism was placing theassembly of the lid and the nucleic acid extraction element 20 into thereaction vessel (reference code 34 in the drawings) in the nucleic acidamplification cartridge, the turning member was turned to close the lidof the reaction vessel, whereby the solid matrix was sealed into thereaction vessel (34 in the drawings) while it was completely submergedin the reaction liquid.

Next, the heat block (reference code 70 in the drawings) of thetemperature controlling mechanism was driven to change the temperatureof the reaction liquid in the reaction vessel, whereby amplification ofthe target nucleic acid was performed. Temperature change was performedin the following pattern: 120 seconds at 95° C.→50 cycles of (4 secondsat 95° C.±60 seconds at 54° C.)→60 seconds at 95° C.→90 seconds at 45°C.

In the SNP typing, Tm analysis was employed. In the Tm analysis,temperature of reaction liquid containing the amplified nucleic acid wasincreased from 45° C. to 95° C. at a rate of 1° C. per 3 seconds, andreal-time fluorescence intensity measurement was performed. Themeasurement was made for two wavelength spectra of: 515 through 555 nm(*2) and 585 through 750 nm (*3). The SNP typing was made for each ofthe wavelengths ((*2), (*3)). Results of the fluorescence intensitymeasurements at each wavelength are given in FIG. 34, with thehorizontal axis representing the temperature, and the vertical axisrepresenting the derivative value (rate of change) of the fluorescenceintensity.

As understood from FIG. 34, in whichever of the wavelengths *2 and *3,the change curve of the derivative value (rate of change) for themeasured fluorescence intensity shows two peaks. These peaks representSNP wild-type and mutation-type, demonstrating that the target nucleicacid was amplified to a satisfying extent where these two types aredifferentiated from each other.

Example 2

In this Example, purification of nucleic acid was performed in the sameway as in Example 1. Amplification was performed by using ICNA method,and then SNP typing was performed. An amplification reagent employed wasCycleave ICAN human ALDH2 Typing Kit (Cat. No. CY101) manufactured byTaKaRa Inc. The amount and composition of the reagent compound liquidsA, B held in the reagent vessels (reference code 32 ₁, 32 ₂ in thedrawings) of the cartridge main body were as shown in Table 3. Theamount pipetted and conditions for mixing these reagent compound liquidsA, B, and the mount of the reaction liquid pipetted were the same as inExample 1. TABLE 3 40 μL Reagent Mix A Sterile Distilled Water 15.2 μL2× ICAN Reaction Buffer 20 μL RNase H 1.6 μL BcaBEST DNA Polymerase 3.2μL Reagent Mix B Sterile Distilled Water 13.6 μL 2× ICAN Reaction Buffer20 μL ALDH2 ICAN Primer Mix 3.2 μL ALDH2 Probe Mix 3.2 μL

(Reaction Conditions)

The reaction was allowed for an hour after the solid matrix wasincubated for 300 seconds at 70° C. while being kept in the reactionliquid. The one-hour reaction was made of 60 cycles, each cycleincluding a first step of 30 seconds without fluorescence intensitymeasurement, and a second step of 30 seconds with fluorescence intensitymeasurement. The fluorescence intensity measurement performed in thesecond step of each cycle was real-time measurement. The measurement wasmade for two wavelengths of 515 through 555 nm (mt) and 585 through 750nm (wt), and SNP typing was made for each of the SNP mutation-type andwild-type. Results of the fluorescence intensity measurements at eachwavelength are given in FIG. 35, with the horizontal axis representingthe number of cycles, and the vertical axis representing thefluorescence intensity.

As understood from FIG. 35, after a lapse of a certain number of cycles,the fluorescence intensity which represents the SNP mutation-typeincreased but the fluorescence intensity which represents the SNPwild-type showed little increase even if the number of cycles wasincreased. Therefore, the result shown in FIG. 35 demonstrates that thetarget nucleic acid was amplified selectively and sufficiently, to anextent where these two types are differentiated from each other.

Example 3

In this Example, purification of nucleic acid was performed in the sameway as in Example 1. Amplification was made by LAMP method, and resultswere tested by SNP typing. An amplification reagent employed was Loopamp P450 typing reagent kit (CYP2C9*3) manufactured by Eiken ChemicalCo., Ltd. The composition of the reagent compound liquids A, B held inthe reagent vessels (reference code 33A, 33B in the drawings) of thecartridge main body were as shown in Table 3. The amount pipetted andconditions for mixing these reagent compound liquids A, B, and the mountof the reaction liquid pipetted were the same as in Example 1. TABLE 440 μL Reagent Mix A Sterile Distilled Water 9.6 μL Reaction Mix. SNP 16μL Fluorescent Detection Reagent for Genome 3.2 μL 10 mM Tris Solution:PH8.0 8 μL Bst DNA Polymerase 3.2 μL Reagent Mix B Sterile DistilledWater 11.2 μL Reaction Mix. SNP 16 μL Primer Mix. for 2C9*3(C) 12.8 μLor Primer Mix. for 2C9*3(A)

(Reaction Conditions)

The reaction was allowed for five minutes at 95° C. and then for an hourat 60° C. with the solid matrix being kept in the reaction liquid. Theone-hour reaction was made of 60 cycles, each cycle including a firststep of 30 seconds without fluorescence intensity measurement, and asecond step of 30 seconds with fluorescence intensity measurement. Thefluorescence intensity measurement performed in the second step of eachcycle was real-time measurement for a wavelength of 515 through 555 nm.Results of the fluorescence intensity measurements at the second step ofeach cycle are given in FIG. 36, with the horizontal axis representingthe number of cycles, and the vertical axis representing thefluorescence intensity.

As understood from FIG. 36, after a lapse of a certain number of cycles,the fluorescence intensity which represents the SNP mutation-type(Allele A in the figure) increased but the fluorescence intensity whichrepresents the SNP wild-type (Allele G in the figure) showed littleincrease even if the number of cycles was increased. Therefore, theresult shown in FIG. 36 demonstrates that the target nucleic acid (SNPwild-type) was amplified selectively and sufficiently, to an extentwhere these two types are differentiated from each other.

As understood from the results of Examples 1 through 3, purification ofnucleic acid performed in the nucleic acid extraction element accordingto the first embodiment of the present invention enables appropriateamplification of a target nucleic acid not only when PCR method was usedbut also when ICAN method or LAMP method was used as the amplificationmethod. As described earlier, ICAN method and LAMP method are notcapable of inducing sufficient amplification reaction if a targetnucleic acid has not been purified to an appropriately high level. Basedon this fact, one can claim that the method of purification described inthe first embodiment according to the present invention is able topurify a target nucleic acid appropriately. Therefore, the purificationmethod described above can be utilized appropriately as a pretreatmentnot only for PCR method but also for other amplification methods, andcontributes to time saving in the amplification.

Also, Examples 1 through 3 demonstrated automatic analysis of nucleicacid by using the nucleic acid purification cartridge, the nucleic acidextraction cartridge and the nucleic acid analyzer according to thefirst embodiment of the present invention. The examples furtherdemonstrated that a variety of known nucleic acid amplification methods,not only limited to PCR method but also including PCR method forexample, can be utilized.

It should be noted here that the discussion on Examples 1 through 3demonstrated that nucleic acid was amplified appropriately based on theconfiguration according to the first embodiment of the presentinvention. It is believed that the configuration according to the secondembodiment of the present invention can also amplify nucleic acidappropriately, and provides the same advantages described above.

1. A nucleic acid extraction container comprising: a nucleic acidextraction element for extraction of a target nucleic acid from a sampleand carrying of the extracted nucleic acid; and a container main bodyformed as a separate member from the nucleic acid extraction element,including a housing vessel for storage of the nucleic acid extractionelement.
 2. The nucleic acid extraction container according to claim 1,wherein the nucleic acid extraction element includes a solid matrix forsupporting the target nucleic acid and a holding member for holding thesolid matrix.
 3. The nucleic acid extraction container according toclaim 2, wherein the solid matrix is held so as to be tilted withrespect to a vertical axis of the holding member.
 4. The nucleic acidextraction container according to claim 3, wherein the solid matrix issupported horizontally or substantially horizontally with respect tosaid vertical axis.
 5. The nucleic acid extraction container accordingto claim 3, wherein the solid matrix is supported by the holding memberso as to be penetrated by the holding member.
 6. The nucleic acidextraction container according to claim 5, wherein the holding memberincludes a tapered portion with a decreasing diameter toward a tip, apin-shaped portion extending from the tapered portion for penetrationthrough the solid matrix, and a holding tab for preventing the solidmatrix from coming off the pin-shaped portion.
 7. The nucleic acidextraction container according to claim 3, wherein the solid matrix isformed as a disc.
 8. The nucleic acid extraction container according toclaim 2, wherein the solid matrix is formed as a sheet and is held so asto be suspended with respect to the holding member.
 9. The nucleic acidextraction container according to claim 8, wherein the holding memberincludes a clip portion for clipping an end of the solid matrix therebysuspending the solid matrix.
 10. The nucleic acid extraction containeraccording to claim 2, wherein the container main body includes one ormore cleansing vessels for holding cleansing liquid necessary to removean extra component from the solid matrix.
 11. The nucleic acidextraction container according to claim 10, wherein the container mainbody is provided with surplus cleansing liquid removal means for removalof surplus cleansing liquid from or around the solid matrix.
 12. Thenucleic acid extraction container according to claim 11, wherein thesurplus cleansing liquid removal means includes a water absorbingmember.
 13. The nucleic acid extraction container according to claim 1,which is configured as a cartridge for setting in a nucleic acidanalyzer.
 14. The nucleic acid extraction container according to claim13, wherein the nucleic acid analyzer includes a carrier member fortaking the nucleic acid extraction element which supports the targetnucleic acid from the housing vessel and moving the nucleic acidextraction element to another location, the nucleic acid extractionelement including an engagement portion for engagement with the carriermember.
 15. The nucleic acid extraction container according to claim 14,wherein the engagement portion is cylindrical for fitting with a tipportion of the carrier member.
 16. The nucleic acid extraction containeraccording to claim 15, wherein the engagement portion includes one ormore cutouts for application of elastic force to the carrier member uponfitting with the tip portion of the carrier member.
 17. The nucleic acidextraction container according to claim 16, wherein the cutout orcutouts include at least any one of a slit, a notch and a through hole.18. The nucleic acid extraction container according to claim 14, whereinthe holding member includes a projection for breaking the fit betweenthe carrier member and the engagement portion.
 19. The nucleic acidextraction container according to claim 18, wherein the projection isprovided by a flange extending outward.
 20. The nucleic acid extractioncontainer according to claim 18, wherein the housing vessel includes astepped portion for seating the projection upon placement of the nucleicacid extraction element in the housing vessel.
 21. A method of removingan extra component other than a target nucleic acid from a solid matrixwhich supports the target nucleic acid, using a cleansing liquid,comprising a repeated cycle of a state in which the solid matrix issubmerged in the cleansing liquid and a state in which the solid matrixis not submerged in the cleansing liquid, caused by moving the solidmatrix in up-and-down directions relatively to the cleansing liquid. 22.The method of cleansing a solid matrix according to claim 21, whereinthe solid matrix is tilted with respect to the up-and-down directions inthe repeated cycle of the state in which the solid matrix is submergedin the cleansing liquid and the state in which the solid matrix is notsubmerged in the cleansing liquid.
 23. The method of cleansing a solidmatrix according to claim 22, wherein the solidmatrix is horizontal orsubstantially horizontal to the moving directions in the repeated cycleof the state in which the solid matrix is submerged in the cleansingliquid and the state in which the solid matrix is not submerged in thecleansing liquid.
 24. The method of cleansing a solid matrix accordingto claim 21, wherein surplus cleansing liquid is removed from the solidmatrix by a water absorbing member after completion of the soaking inthe cleansing liquid.
 25. A method of purifying a target nucleic acidusing a nucleic acid extraction element which includes a solid matrixfor supporting the target nucleic acid and a holding member for holdingthe solid matrix, comprising: a nucleic acid supporting step of causingthe solid matrix to support the target nucleic acid in a sample bysoaking the solid matrix in the sample; and a cleansing step of removingan extra component other than the target nucleic acid from the solidmatrix, using a cleansing liquid; wherein the cleansing step isperformed by a repeated cycle of a state in which the solid matrix issubmerged in the cleansing liquid and a state in which the solid matrixis not submerged in the cleansing liquid, caused by moving the solidmatrix in up-and-down directions relatively to the cleansing liquid. 26.The method of purifying a target nucleic acid according to claim 25,wherein the solid matrix held by the holding member is tilted withrespect to a vertical axis of the holding member in the nucleic acidextraction element, the cleansing step being performed by moving theextraction element in the up-and-down directions, with the solid matrixtilted with respect to the up-and-down directions.
 27. The method ofpurifying a target nucleic acid according to claim 26, wherein thecleansing step is performed by moving the nucleic acid extractionelement in the up-and-down directions, with the solid matrix beingperpendicular or substantially perpendicular to the up-and-downdirections.
 28. The method of purifying a target nucleic acid accordingto claim 25, wherein the cleansing step includes removal of surpluscleansing liquid from the solid matrix by a water absorbing member aftercompletion of the repeated soaking of the solid matrix in the cleansingliquid.
 29. A mechanism for removing an extra component other than atarget nucleic acid from a solid matrix which supports the targetnucleic acid, using a cleansing liquid, wherein the mechanism repeats acycle of a state in which the solid matrix is submerged in the cleansingliquid and a state in which the solid matrix is not submerged in thecleansing liquid, by moving the solid matrix in up-and-down directionsrelatively to the cleansing liquid.
 30. The cleansing mechanism for asolid matrix according to claim 29, wherein the solid matrix is tiltedwith respect to the up-and-down directions when moved in the up-and-downdirections.
 31. The cleansing mechanism for a solid matrix according toclaim 30, wherein the solid matrix is perpendicular or substantiallyperpendicular to the up-and-down directions when moved in theup-and-down directions.